Polymorphism of the human C3b/C4b receptor. Identification of a third allele and analysis of receptor phenotypes in families and patients with systemic lupus erythematosus.
We have isolated C3bR from surface-labeled erythrocytes of 180 normal individuals and 45 patients with SLE. These studies have identified a previously unrecognized C3bR molecule on E with a Mr of approximately 160,000 daltons on nonreduced SDS-polyacrylamide gels. A similar receptor phenotype is also found on other C3bR-bearing peripheral blood leukocytes. Family studies demonstrate that this approximately 160,000-dalton molecule represents a third allele that is inherited in a codominant fashion at the same locus as the two previously described C3bR alleles. In unrelated normal donors a common allele (A) determines an approximately 190,000-dalton C3bR (gene frequency 0.83), a second allele (B) determines an approximately 220,000-dalton C3bR (gene frequency = 0.16), and a third rare allele (C) determines an approximately 160,000-dalton C3bR (gene frequency = 0.01). There were no major differences in gene frequencies among Caucasians and blacks or normal individuals and patients with SLE. However, compared with normal individuals, heterozygous C3bR-AC patients with SLE express large amounts of the approximately 160,000-dalton C3bR on E. Expression of C3bR molecules among heterozygous siblings is similar, suggesting that an inherited factor controls expression of the two molecules in heterozygous donors. These observations constitute an instructive example of a structural polymorphism of an integral membrane glycoprotein and provide a structural and genetic basis for further molecular and functional analyses of C3bR in normal and patient populations.


Injectable microbubbles as contrast agents for diagnostic ultrasound imaging: the key role of perfluorochemicals.
Ultrasonography has, until recently, lacked effective contrast-enhancing agents. Micrometer-sized gas bubbles that resonate at a diagnostic frequency are ideal reflectors for ultrasound. However, simple air bubbles, when injected into the blood stream, disappear within seconds through the combined effects of Laplace pressure, blood pressure, and exposure to ultrasound energy. Use of fluorocarbon vapor, by extending the persistence of microbubbles in vivo from seconds to minutes, propelled contrast ultrasonography into clinical practice. Imaging techniques that selectively suppress tissue, but not microbubble signal, further increase image contrast. Approved products consist of C3F8 or SF6 microbubbles, and N2 microbubbles osmotically stabilized with C6F14. These agents allow the detection and characterization of cardiovascular abnormalities and solid organ lesions, such as tumors. By providing higher quality images, they improve the accuracy and confidence of disease diagnosis, and can play a decisive role in clinical decision making. New objectives include agents that target specific cells for the molecular imaging of disease, and drug and gene delivery, including ultrasound-triggered delivery.


Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study.
TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers - likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.


Eyeing up the Future of the Pupillary Light Reflex in Neurodiagnostics.
The pupillary light reflex (PLR) describes the constriction and subsequent dilation of the pupil in response to light as a result of the antagonistic actions of the iris sphincter and dilator muscles. Since these muscles are innervated by the parasympathetic and sympathetic nervous systems, respectively, different parameters of the PLR can be used as indicators for either sympathetic or parasympathetic modulation. Thus, the PLR provides an important metric of autonomic nervous system function that has been exploited for a wide range of clinical applications. Measurement of the PLR using dynamic pupillometry is now an established quantitative, non-invasive tool in assessment of traumatic head injuries. This review examines the more recent application of dynamic pupillometry as a diagnostic tool for a wide range of clinical conditions, varying from neurodegenerative disease to exposure to toxic chemicals, as well as its potential in the non-invasive diagnosis of infectious disease.


Power distribution across the face of different light guides and its effect on composite surface microhardness.
The aim of this study was to examine the influence of light guide type on the distribution of irradiant emission from a light-emitting diode (LED) curing light and to measure the effect of light dispersion on surface microhardness across the top and bottom surfaces of two types of composite resins. A laser beam analyzer (LBA-700, Spiricon) was used to evaluate light distribution (Top Hat factor [THF]) across the distal surface of a standard and turbo light guide from an LED curing light (Bluephase, Ivoclar Vivadent). Composites (Z100 [hybrid], A110 [microfill]; 3M ESPE) were placed in blackened rings (2 x 11 mm) and exposed at 0 mm for 5 seconds (Z100) or 15 seconds (A110) using the light guides at similar irradiance, energy density, and exit diameters (N = 5). Similar irradiance values were produced by using the turbo light guide on the "low power" setting of the curing light and the "high power" setting when using the standard light guide. THF values were analyzed with an unpaired t-test. Knoop hardness (KHN) was determined on the top and bottom surfaces (Leco) in 1-mm lateral increments from the specimen center and proceeding 4 mm in both east-west and north-south directions. The effects of the major factors (light guide type and lateral distance) on the hardness of each composite were analyzed using multiple analysis of variance (ANOVA), and a two-tailed, unpaired Dunnett's t-test determined when lateral hardness values significantly differed from that at the specimen center. The percentage difference between maximum and minimum (max-min) hardness values for each specimen, with respect to distance from specimen center, and the percentage decrease for the standard and turbo light guides, with respect to both composite resin types, were compared using ANOVA and the Tukey's post-hoc test. The standard guide had a significantly higher (i.e., more uniform light distribution) THF than did the turbo tip (p < 0.001). For the microfill, significant differences in hardness were found based on the distance from the specimen center (p < 0.0001), and with respect to the top or bottom surfaces (p < 0.0001). However, no difference was found between the two types of light guides (p = 0.939). For the hybrid, significant differences in hardness were found based on lateral distance (p < 0.001), surface (p < 0.001), and light guide type (p = 0.045).However, for both composites, significant interactions were present. Significant differences were found between hardness at the specimen center and at various lateral distances, depending on composite type, surface, and light guide type. The percent age max-min hardness decrease across the surface was significantly less for the standard light guide (p < 0.0001) and at the top surface (p < 0.02) with both composite resin types. Also, the least percentage max-min hardness decrease occurred on the top surface of the microfill material (p < 0.001). The standard light guide produced a more homogeneous distribution of light across the tip end compared with the turbo light guide, based on the THF. Composite surface hardness patterns correlated with the applied irradiance distribution profiles, yielding greater hardness at higher irradiance locations for both top and bottom surfaces. Light guide selection may influence the uniformity of surface and subsurface hardness when light-curing composite resin restorations.


New Records of Species of Macrovalvitrematidae Yamaguti, 1963 (Monogenea) from Puerto Rico Including One New Genus and Two New Species.
New records of Macrovalvitrematidae Yamaguti, 1963 are reported from Puerto Rico including Buccamagna archosarga n. gen., n. sp. from the western Atlantic sea bream Archosargus rhomboidalis, Pseudotagia pomadasys n. sp. from the roughneck grunt Pomadasys corvinaeformis, and new locality records for the species Neopterinotrematoides avaginata Suriano, 1975 and Macrovalvitrema argentinensis (Suriano, 1975) n. comb. from the whitemouth croaker Micropogonias furnieri . Buccamagna n. gen. is based on the anterior end and the oral disc being much larger and wider than width of the body proper, by having a pair of rectangular and sclerotized oral suckers, by lacking a pharynx, by having an excessive narrowness in the proximal area of both valves, and by having both valves of the 3 pairs of large clamps armed with several accessory sclerites. Pseudotagia pomadasys n. sp. is distinguished by the absence of serration of lateral sclerites and the relative size of the clamps, having the 2 larger pairs positioned centrally in the haptor and the 2 smaller pairs in the outer positions. Macrovalvitrematoides and Neomacrovalvitrema are synonymized with Macrovalvitrema, which is amended to include Macrovalvitrema micropogoni (Pearse, 1949) n. comb. and Macrovalvitrema argentinensis (Suriano, 1975) n. comb. based on recognition of identical clamp morphologies. In this study the clamp is emphasized as the key taxonomic character for macrovalvitrematid genera. Except for Pseudotagia, the Macrovalvitrematidae have distinctively longer than wide clamps (at least twice as long as wide); with the dorsal valve larger and with a more-complex skeletal structure (g1, g2, i, k, and f sclerites) than ventral valve (c and d sclerites); and 2 clamp morphologies. Characteristics not previously described but present in all macrovalvitrematid monogeneans except Pseudotagia included: (1) asymmetrical plate (plate b) along the ventral valve with a thin fissure on one side, and (2) mirror image arrangement of the pairs of clamps.


Efficacy of plastic pot scrubbers as a replacement for roughage in high-concentrate cattle diets.
Effects of plastic pot scrubbers on feedlot performance and ruminal metabolism of steers fed all-concentrate diets were determined. In Trial 1, 31 crossbred steers (means initial BW, 290 kg) were penned and fed individually. Treatments were 1) 85% concentrate-15% corn silage diet, 2) 100% concentrate diet, and 3) 100% concentrate diet + ruminal insertion of eight plastic pot scrubbers per steer. During the first 112 d of the trial, steer ADG did not differ (P greater than .10) due to treatment. From d 113 to 152, steers provided with pot scrubbers had 16% greater ADG than those fed the 100% concentrate diet without pot scrubbers (P = .18). In Trial 2, 78 crossbred steers (means initial BW, 315 kg) were penned individually and fed the diets used in Trial 1. Steers fed the 100% concentrate diet received zero, four or eight pot scrubbers. From d 113 to 167, steers provided with four or eight pot scrubbers or fed the 85% concentrate diet had greater (P less than .10) gains than steers fed the 100% concentrate diet without pot scrubbers. In Trial 3, 120 steers (means initial BW, 286 kg) were grouped in 12 pens and limit-fed an all-concentrate diet for 84 d. Sixty steers were provided with six pot scrubbers each. Performance was not affected (P greater than .10) by the use of pot scrubbers during the 84-d growing phase. During the subsequent 84-d finishing phase, half the steers receiving each treatment were switched to either an 85% concentrate-15% corn silage or an 100% concentrate diet offered for ad libitum consumption.(ABSTRACT TRUNCATED AT 250 WORDS)


A gene responsible for the pigment dispersion syndrome maps to chromosome 7q35-q36.
To demonstrate the inheritance of the pigment dispersion syndrome in 4 families and to determine the location of a gene responsible for this syndrome. Fifty-four members of 4 families affected by the pigment dispersion syndrome and pigmentary glaucoma. All 4 families are white. Two of the pedigrees are of Irish descent, and 2 are of mixed western European descent that includes some Irish ancestry. Individuals from 4 pedigrees affected by the pigment dispersion syndrome and their spouses were clinically examined for evidence of the pigment dispersion syndrome. DNA samples from patients and appropriate family members were used for a genome screen using microsatellite repeat markers distributed throughout the human genome. Genotypes were used for linkage analysis to identify markers segregating with the disease trait. Twenty-eight patients showed clinical evidence of the pigment dispersion syndrome. Of these, 14 also had elevated intraocular pressures requiring medical or surgical treatment or both. Significant linkage was observed between the disease phenotype and markers located on the telomere of the long arm of human chromosome 7 (i.e., 7q35-q36). The maximum 2-point lod score (i.e., Zmax) for a single pedigree (i.e., PDS5) was 5.72 at theta = 0 for markers D7S2546 and D7S550. An analysis of affected recombinant individuals demonstrated that the responsible gene is located in a 10-centimorgan interval between markers D7S2462 and D7S2423. The pigment dispersion syndrome was found to be inherited as an autosomal dominant trait in 4 affected pedigrees. The gene responsible for the syndrome in these 4 families maps to the telomeric end of the long arm of chromosome 7 (i.e., 7q35-q36). Locating a gene responsible for this condition is the first step toward the isolation of the gene itself. Characterization of the responsible gene will help elucidate the pathophysiology of this disease and potentially will lead to new methods of diagnosis and treatment.


Comparison of speed of sound ultrasound with single photon absorptiometry for determining fracture odds ratios.
The purpose of this study was to compare the ability of ultrasound velocity measurement with that of single photon absorptiometry to determine the odds of history of fracture since age 40, for a population-based study. To do this we computed odds ratios and 95% confidence intervals for 809 women and 502 men, aged 50 years and older, who are participants in the prospective phase of the Saunders County Bone Quality Study. These participants received both the ultrasound and single photon absorptiometry bone measurements at the initiation of the study. In addition, a history of all fractures that had occurred to participants since age 40 was obtained. The two bone assessment methods were compared by examining the magnitude of the odds ratios, to determine which produces the highest estimate of the probability of odds of fracture, and by examining widths of the respective confidence intervals to show which estimate of odds ratio is the most precise. Ultrasound velocity estimates a higher probability of odds of both low-trauma fractures and all fractures than distal radius and ulna bone mineral content, but lower than bone mineral density at the same sites for both women and men. However, the ultrasound measure is more precise than bone mineral density, but less precise than bone mineral content. We conclude that ultrasound velocity is as good as single photon absorptiometry in estimating odds of fracture.


What can studying musicians tell us about motor control of the hand?
Most standard accounts of human anatomy and physiology are designed to meet the requirements of medical education and therefore consider their subject matter from the standpoint of typical rather than outstanding levels of performance. To understand how high levels of skill are developed and maintained, it is necessary to study elite groups such as professional athletes or musicians. This can lead to the rediscovery of arcane knowledge that has fallen into neglect through a lack of appreciation of its significance. For example, although variability in the muscles and tendons of the hand was well known in the nineteenth and early twentieth centuries, it is through recent studies of musicians that its practical significance has become better appreciated. From even a cursory acquaintance with the training methods of sportsmen and women, dancers and musicians, it is clear that sophisticated motor skills are developed only at the cost of a great deal of time and effort. Over a lifetime of performance, musicians arguably spend more time in skill acquisition than almost any other group and offer a number of unique advantages for the study of motor control. Such intensive training not only modifies cortical maps but may even affect the gross morphology of the central nervous system. There is also evidence that in certain individuals this process can become maladaptive. Recent studies of musicians suggest that intensive training can lead to the appearance of ambiguities in the cortical somatosensory representation of the hand that may be associated with the development of focal dystonia; a condition to which musicians are particularly prone. The realization that changes in cortical maps may underlie dystonia has led to the development of new approaches to its treatment, which may ultimately benefit musicians and non-musicians alike.


Effects of parietal TMS on somatosensory judgments challenge interhemispheric rivalry accounts.
Interplay between the cerebral hemispheres is vital for coordinating perception and behavior. One influential account holds that the hemispheres engage in rivalry, each inhibiting the other. In the somatosensory domain, a seminal paper claimed to demonstrate such interhemispheric rivalry, reporting improved tactile detection sensitivity on the right hand after transcranial magnetic stimulation (TMS) to the right parietal lobe (Seyal, Ro, & Rafal, 1995). Such improvement in tactile detection ipsilateral to TMS could follow from interhemispheric rivalry, if one assumes that TMS disrupted cortical processing under the coil and thereby released the other hemisphere from inhibition. Here we extended the study by Seyal et al. (1995) to determine the effects of right parietal TMS on tactile processing for either hand, rather than only the ipsilateral hand. We performed two experiments applying TMS in the context of median-nerve stimulation; one experiment required somatosensory detection, the second somatosensory intensity discrimination. We found different TMS effects on detection versus discrimination, but neither set of results followed the prediction from hemispheric rivalry that enhanced performance for one hand should invariably be associated with impaired performance for the other hand, and vice-versa. Our results argue against a strict rivalry interpretation, instead suggesting that parietal TMS can provide a pedestal-like increment in somatosensory response.


Effects of fossil fuel and total anthropogenic emission removal on public health and climate.
Anthropogenic greenhouse gases and aerosols are associated with climate change and human health risks. We used a global model to estimate the climate and public health outcomes attributable to fossil fuel use, indicating the potential benefits of a phaseout. We show that it can avoid an excess mortality rate of 3.61 (2.96-4.21) million per year from outdoor air pollution worldwide. This could be up to 5.55 (4.52-6.52) million per year by additionally controlling nonfossil anthropogenic sources. Globally, fossil-fuel-related emissions account for about 65% of the excess mortality, and 70% of the climate cooling by anthropogenic aerosols. The chemical influence of air pollution on aeolian dust contributes to the aerosol cooling. Because aerosols affect the hydrologic cycle, removing the anthropogenic emissions in the model increases rainfall by 10-70% over densely populated regions in India and 10-30% over northern China, and by 10-40% over Central America, West Africa, and the drought-prone Sahel, thus contributing to water and food security. Since aerosols mask the anthropogenic rise in global temperature, removing fossil-fuel-generated particles liberates 0.51(±0.03) °C and all pollution particles 0.73(±0.03) °C warming, reaching around 2 °C over North America and Northeast Asia. The steep temperature increase from removing aerosols can be moderated to about 0.36(±0.06) °C globally by the simultaneous reduction of tropospheric ozone and methane. We conclude that a rapid phaseout of fossil-fuel-related emissions and major reductions of other anthropogenic sources are needed to save millions of lives, restore aerosol-perturbed rainfall patterns, and limit global warming to 2 °C.


An efficient method for isolating antibody fragments against small peptides by antibody phage display.
We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of αs1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do not always bind efficiently to passive adsorption surfaces, and we developed a simple method to quantify the binding capacity of surfaces with the peptides. Background binding (the binding of scFvs to the background matrix) is an obstacle for successful selection, and we evaluated two methods that drastically reduced the background binding. An optimized method therefore enabled a panning procedure where the specific (peptide binding) scFv-phages were always dominant. Using 15-mer peptides immobilized on Nunc Immobilizer Streptavidin plates, we successfully generated scFvs specifically against them. The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides.


Computational toxicology using the OpenTox application programming interface and Bioclipse.
Toxicity is a complex phenomenon involving the potential adverse effect on a range of biological functions. Predicting toxicity involves using a combination of experimental data (endpoints) and computational methods to generate a set of predictive models. Such models rely strongly on being able to integrate information from many sources. The required integration of biological and chemical information sources requires, however, a common language to express our knowledge ontologically, and interoperating services to build reliable predictive toxicology applications. This article describes progress in extending the integrative bio- and cheminformatics platform Bioclipse to interoperate with OpenTox, a semantic web framework which supports open data exchange and toxicology model building. The Bioclipse workbench environment enables functionality from OpenTox web services and easy access to OpenTox resources for evaluating toxicity properties of query molecules. Relevant cases and interfaces based on ten neurotoxins are described to demonstrate the capabilities provided to the user. The integration takes advantage of semantic web technologies, thereby providing an open and simplifying communication standard. Additionally, the use of ontologies ensures proper interoperation and reliable integration of toxicity information from both experimental and computational sources. A novel computational toxicity assessment platform was generated from integration of two open science platforms related to toxicology: Bioclipse, that combines a rich scriptable and graphical workbench environment for integration of diverse sets of information sources, and OpenTox, a platform for interoperable toxicology data and computational services. The combination provides improved reliability and operability for handling large data sets by the use of the Open Standards from the OpenTox Application Programming Interface. This enables simultaneous access to a variety of distributed predictive toxicology databases, and algorithm and model resources, taking advantage of the Bioclipse workbench handling the technical layers.


Basics of genome-scale metabolic modeling and applications on C1-utilization.
It is fundamental to understand the relationship between genotype and phenotype in biology. This requires comprehensive knowledge of metabolic pathways, genetic information and well-defined mathematic modeling. Integration of knowledge on metabolism with mathematical modeling results in genome-scale metabolic models which have proven useful to investigate bacterial metabolism and to engineer bacterial strains capable of producing value-added biochemical. Single carbon substrates such as methane and carbon monoxide have drawn interests and they assumed one of next-generation feedstocks because of their high abundance and low price. The methylotroph and acetogen-based biorefineries hold promises for bioconversion of C1 substrates into biofuels and high value compounds. As an effort on expanding our knowledge on C1 utilization approaches, in silico computational framework of C1-metabolism in methylotrophic and acetogenic bacteria has been developed. In this review, genome-scale metabolic models for C1-utilizing bacteria and well-established analysis tools are presented for potential uses for study of C1 metabolism at the genome scale and its application in metabolic engineering.


NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses.
NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8+ T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8+ T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity in vivo impairs DC cross-presentation and CD8+ T cell activation. DCs from Crohn's patients expressing NOD2 polymorphisms show dysregulated cross-presentation and CD8+ T cell responses. Our findings reveal unidentified mechanisms that underlie CD8+ T cell responses to bacteria in health and in Crohn's.


Molecular selectivity and immiscibility during the crystallization of mixtures of a set of homologous self-assembling molecules.
Utilizing self-assembly to create supramolecular structures is an active area at this time. Hybrid materials created by blending or doping, e.g., organic/inorganic or donor/acceptor complexes are of great interest in the design of novel materials systems. The effect of mixing of any two self-assembling molecules to modify the properties and to understand if the process of blending changes the nature of the self-assembly would be of interest. We discuss here the effect of blending of two (hydrogen bond mediated) self-assembling homologous molecules on the structure and morphology. Materials that are candidate vehicles for phase-change inkjet technology, biscarbamates with alkyl side chains, are chosen for this study. Thermal analysis and IR spectra indicate that, when two biscarbamates differing only in the length of the alkyl chain are blended, the two components are immiscible, although they are chemically similar. There is no intercalation of the alkyl chains and cocrystallization. They are thus an example of a self-sorting system. The extent of hydrogen bonding and the packing of the alkyl chains are not affected. However, each serve as a nucleating agent and reduce the size of the spherulites and crystallinity. The spherulitic growth rate decreases upon blending. Partial melting experiments show that the spherulites of each component do not form independently, but are intermixed, implying that one acts as the nucleating sites for the other. Thus, although these are self-sorting, the components in the mixture affect the morphology of each other upon crystallization. The behavior of this small molecule mixture is compared with those of hydrogen-bonded polymer blends. Studies of this nature on blends of self-assembling molecules are expected to be important in materials design for optimizing properties.


High Levels of Canonical Wnt Signaling Lead to Loss of Stemness and Increased Differentiation in Hematopoietic Stem Cells.
Canonical Wnt signaling regulates the self-renewal of most if not all stem cell systems. In the blood system, the role of Wnt signaling has been the subject of much debate but there is consensus that high Wnt signals lead to loss of reconstituting capacity. To better understand this phenomenon, we have taken advantage of a series of hypomorphic mutant Apc alleles resulting in a broad range of Wnt dosages in hematopoietic stem cells (HSCs) and performed whole-genome gene expression analyses. Gene expression profiling and functional studies show that HSCs with APC mutations lead to high Wnt levels, enhanced differentiation, and diminished proliferation but have no effect on apoptosis, collectively leading to loss of stemness. Thus, we provide mechanistic insight into the role of APC mutations and Wnt signaling in HSC biology. As Wnt signals are explored in various in vivo and ex vivo expansion protocols for HSCs, our findings also have clinical ramifications.


Atp6ap2/(pro)renin receptor interacts with Par3 as a cell polarity determinant required for laminar formation during retinal development in mice.
(Pro)renin receptor [(P)RR], also known as Atp6ap2, has attracted growing attention as a key molecule for tissue renin-angiotensin system (RAS). In addition to its role in tissue RAS activation, Atp6ap2/(P)RR was originally identified as an accessory subunit for vacuolar H(+)-ATPase (v-ATPase), which is a multisubunit proton pump involved in diverse and fundamental cellular physiology. In this study, to elucidate the physiological function of Atp6ap2/(P)RR during retinal development in mammals, we used Cre-LoxP system to generate photoreceptor-specific conditional knock-out (CKO) mice, and revealed a critical role of Atp6ap2/(P)RR in photoreceptor development. Deletion of photoreceptor Atp6ap2/(P)RR did not affect retinal cell differentiation, but led to laminar disorganization around the outer nuclear layer together with severe dysfunction of photoreceptor cells. In the CKO mice, cell adhesion and polarity molecules, some of which were colocalized with Atp6ap2/(P)RR at the apical edge of the wild-type developing retina, were substantially dispersed together with mislocalization of retinal progenitor cells apart from the apical surface. Among theses molecules, coimmunoprecipitation using retinal homogenates and ATP6AP2/(P)RR-transfected cells showed that Atp6ap2/(P)RR interacted with partitioning defective 3 homolog (PAR3) protein, which is known to function in the Par-atypical protein kinase C (aPKC) system. Furthermore, yeast two-hybrid assays demonstrated direct molecular interaction between ATP6AP2/(P)RR and PAR3. Our present data revealed the novel function of Atp6ap2/(P)RR required for laminar formation during retinal development. We propose that this cellular activity associated with the Par-aPKC system, in addition to the v-ATPase function and tissue RAS activation, is the third biological role of Atp6ap2/(P)RR.


The functional outcomes of total knee arthroplasty.
As an elective procedure, total knee arthroplasty is under scrutiny to evaluate its cost-effectiveness. In this review, we examined the available literature on total knee arthroplasty to assess the evidence regarding factors associated with better functional outcomes. A structured literature search of English-language databases was performed to identify studies of the functional outcomes of total knee arthroplasty that had been published between 1995 and April 2003. Inclusion criteria were a study of primary total knee arthroplasty, more than 100 knees in the study, provision of baseline data and rating of postoperative outcomes with a standardized symptom scale, and an experimental or quasi-experimental study design. The abstracting form included a list of potential prognostic factors, including comorbidities, radiographic evidence of joint destruction, bone loss, integrity of the extensor mechanism, range of motion, alignment, tibiofemoral angle, and ligament integrity, as well as the characteristics of the operating surgeon, such as procedure volume and experience. Sixty-two studies met the criteria and were reviewed. Total knee arthroplasty was found to be associated with substantial functional improvement, with the effect sizes varying with the measure that was used. Physician-derived measures showed effect sizes of 2.35 and 3.91, whereas patient-derived measures showed smaller effect sizes (1.27 and 1.62). Few investigators used multivariate models to identify associations between outcomes and patient characteristics. Total knee arthroplasty is a generally effective procedure, but the current English-language literature does not support specific recommendations about which patients are most likely to benefit from it.


Trends in the prevalence, incidence and surgical management of carpal tunnel syndrome between 1993 and 2013: an observational analysis of UK primary care records.
To describe the prevalence, incidence and surgical management of carpal tunnel syndrome (CTS), between 1993 and 2013, as recorded in the Clinical Practice Research Datalink (CPRD). We completed a series of cross-sectional epidemiological analyses to observe trends over time. Primary care data collected between 1993 and 2013, stored in the CPRD. Individuals aged ≥18 years were selected. Prevalent and incident episodes of CTS and episodes of surgical intervention were identified using a list of preidentified Read codes. We defined incident episodes as those with no preceding diagnostic code for CTS in the past 2 years of data. Episodes of surgery were expressed as a percentage of the prevalent population during the same calendar year. Joinpoint regression was used to determine significant changes in the underlying trend. The prevalence of CTS increased over the study period, with a particular incline between 2000 and 2004 (annual percentage change 7.81). The female-to-male prevalence ratio reduced over time from 2.74 in 1993 to 1.93 in 2013. The median age of females and males with CTS were noted to increase from 49 and 53 years, respectively in 1993 to 54 and 59 years, respectively in 2013. Incidence was also noted to increase over time. After an initial increase between 1993 and 2007, the percentage of prevalent patients with a coded surgical episode began to decrease after 2007 to 27.41% in 2013 (annual percentage change -1.7). This study has demonstrated that the prevalence and incidence of CTS increased over the study period between 1993 and 2013. Rates of surgery for CTS also increased over the study period; however after 2007, the per cent of patients receiving surgery showed a statistically significant decline back to the rate seen in 2004.


Is the yeast a relevant model for aging of multicellular organisms? An insight from the total lifespan of Saccharomyces cerevisiae.
The applicability of the free radical theory of aging to the yeast S. cerevisiae is a matter of debate. In order to get an insight into this question, we studied the reproductive potential (the number of buds produced), reproductive lifespan (the time during which a yeast cell is able to divide), postreproductive lifespan (duration of life of yeast cells which ceased to divide) and total lifespan (sum of reproductive lifespan and postreproductive lifespan) of three isogenic pairs of yeast strains. Each pair contained a parent strain and a disruptant of gene(s) coding for important antioxidant enzyme(s) (CuZn-superoxide dismutase, all five peroxiredoxins or glutaredoxin 5). Although the reproductive potential was decreased in all antioxidant enzyme-deficient mutants, the differences in the reproductive lifespan between the parent strains and the mutants were less pronounced while postreproductive lifespan and total lifespan were not diminished in the mutants. These results suggest that either the free-radical theory of aging is not applicable to S. cerevisiae or that this yeast is not a proper model organism for the study of aging of higher organisms. In our opinion the latter possibility is more apparent and the increase in cell volume (unavoidable for a cell propagating by budding) rather than accumulation of oxidative damage may be the main reason for the cessation of budding (and perhaps postreproductive death) in S. cerevisiae.


Polymerase chaperoning and multiple ATPase sites enable the E. coli DNA polymerase III holoenzyme to rapidly form initiation complexes.
Cellular replicases include three subassemblies: a DNA polymerase, a sliding clamp processivity factor, and a clamp loader complex. The Escherichia coli clamp loader is the DnaX complex (DnaX(3)δδ'χψ), where DnaX occurs either as τ or as the shorter γ that arises by translational frameshifting. Complexes composed of either form of DnaX are fully active clamp loaders, but τ confers important replicase functions including chaperoning the polymerase to the newly loaded clamp to form an initiation complex for processive replication. The kinetics of initiation complex formation were explored for DnaX complexes reconstituted with varying τ and γ stoichiometries, revealing that τ-mediated polymerase chaperoning accelerates initiation complex formation by 100-fold. Analyzing DnaX complexes containing one or more K51E variant DnaX subunits demonstrated that only one active ATP binding site is required to form initiation complexes, but the two additional sites increase the rate by ca 1000-fold. For τ-containing complexes, the ATP analogue ATPγS was found to support initiation complex formation at 1/1000th the rate with ATP. In contrast to previous models that proposed ATPγS drives hydrolysis-independent initiation complex formation by τ-containing complexes, the rate and stoichiometry of ATPγS hydrolysis coincide with those for initiation complex formation. These results show that although one ATPase site is sufficient for initiation complex formation, the combination of polymerase chaperoning and the binding and hydrolysis of three ATPs dramatically accelerates initiation complex formation to a rate constant (25-50 s(-1)) compatible with double-stranded DNA replication.


Influence of shear stress in perfusion bioreactor cultures for the development of three-dimensional bone tissue constructs: a review.
Bone tissue engineering aims to generate clinically applicable bone graft substitutes in an effort to ease the demands and reduce the potential risks associated with traditional autograft and allograft bone replacement procedures. Biomechanical stimuli play an important role under physiologically relevant conditions in the normal formation, development, and homeostasis of bone tissue--predominantly, strain (predicted levels in vivo for humans <2000 με) caused by physical deformation, and fluid shear stress (0.8-3 Pa), generated by interstitial fluid movement through lacunae caused by compression and tension under loading. Therefore, in vitro bone tissue cultivation strategies seek to incorporate biochemical stimuli in an effort to create more physiologically relevant constructs for grafting. This review is focused on collating information pertaining to the relationship between fluid shear stress, cellular deformation, and osteogenic differentiation, providing further insight into the optimal culture conditions for the creation of bone tissue substitutes.


Botryosphaeriaceae from Eucalyptus plantations and adjacent plants in China.
The Botryosphaeriaceae is a species-rich family that includes pathogens of a wide variety of plants, including species of Eucalyptus. Recently, during disease surveys in China, diseased samples associated with species of Botryosphaeriaceae were collected from plantation Eucalyptus and other plants, including Cunninghamina lanceolata, Dimocarpus longan, Melastoma sanguineum and Phoenix hanceana, which were growing adjacent to Eucalyptus. In addition, few samples from Araucaria cunninghamii and Cedrus deodara in two gardens were also included in this study. Disease symptoms observed mainly included stem canker, shoot and twig blight. In this study, 105 isolates of Botryosphaeriaceae were collected from six provinces, of which 81 isolates were from Eucalyptus trees. These isolates were identified based on comparisons of the DNA sequences of the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS), and partial translation elongation factor 1-alpha (tef1), β-tubulin (tub), DNA-directed RNA polymerase II subunit (rpb2) and calmodulin (cmdA) genes, the nuclear ribosomal large subunit (LSU) and the nuclear ribosomal small subunit (SSU), and combined with their morphological characteristics. Results showed that these isolates represent 12 species of Botryosphaeriaceae, including Botryosphaeria fusispora, Cophinforma atrovirens, Lasiodiplodia brasiliense, L. pseudotheobromae, L. theobromae and Neofusicoccum parvum, and six previously undescribed species of Botryosphaeria and Neofusicoccum, namely B. pseudoramosa sp. nov., B. qingyuanensis sp. nov., B. wangensis sp. nov., N. hongkongense sp. nov., N. microconidium sp. nov. and N. sinoeucalypti sp. nov. Aside from B. wangensis, C. atrovirens and N. hongkongense, the other nine Botryosphaeriaceae species were isolated from Eucalyptus trees in South China. Botryosphaeria fusispora (26 % of the isolates from Eucalyptus) is the dominant species, followed by L. pseudotheobromae (23 % of the isolates from Eucalyptus). In addition to species found on Eucalyptus trees, we also found B. pseudoramosa on M. sanguineum; B. wangensis on C. deodara; C. atrovirens on D. longan; L. theobromae on C. lanceolata, D. longan and P. hanceana; and N. hongkongense on A. cunninghamii. Pathogenicity tests showed that the 12 species of Botryosphaeriaceae are pathogenic to three Eucalyptus clones and that Lasiodiplodia species are the most aggressive. The results of our study suggest that many more species of the Botryosphaeriaceae remain to be discovered in China. This study also provides confirmation for the wide host range of Botryosphaeriaceae species on different plants.


Modeling conduction delays in the corpus callosum using MRI-measured g-ratio.
Conduction of action potentials along myelinated axons is affected by their structural features, such as the axonal g-ratio, the ratio between the inner and outer diameters of the myelin sheath surrounding the axon. The effect of g-ratio variance on conduction properties has been quantitatively evaluated using single-axon models. It has recently become possible to estimate a g-ratio weighted measurement in vivo using quantitative MRI. Nevertheless, it is still unclear whether the variance in the g-ratio in the healthy human brain leads to significant differences in conduction velocity. In this work we tested whether the g-ratio MRI measurement can be used to predict conduction delays in the corpus callosum. We present a comprehensive framework in which the structural properties of fibers (i.e. length and g-ratio, measured using MRI), are incorporated in a biophysical model of axon conduction, to model conduction delays of long-range white matter fibers. We applied this framework to the corpus callosum, and found conduction delay estimates that are compatible with previously estimated values of conduction delays. We account for the variance in the velocity given the axon diameter distribution in the splenium, mid-body and genu, to further compare the fibers within the corpus callosum. Conduction delays have been suggested to increase with age. Therefore, we investigated whether there are differences in the g-ratio and the fiber length between young and old adults, and whether this leads to a difference in conduction speed and delays. We found very small differences between the predicted delays of the two groups in the motor fibers of the corpus callosum. We also found that the motor fibers of the corpus callosum have the fastest conduction estimates. Using the axon diameter distributions, we found that the occipital fibers have the slowest estimations, while the frontal and motor fiber tracts have similar estimates. Our study provides a framework for predicting conduction latencies in vivo. The framework could have major implications for future studies of white matter diseases and large range network computations. Our results highlight the need for improving additional in vivo measurements of white matter microstructure.


Pelvic relapse following subtotal lymphoid irradiation in early stage Hodgkin's disease--an analysis of risk, management, and outcome.
To evaluate the time of onset, method of identification, management, and outcome of pelvic relapse following subtotal lymphoid irradiation (STLI) alone (mantle and paraaortic/spleen or splenic pedicle fields, excluding the pelvis) in supradiaphragmatic Stage I-II Hodgkin's disease. A retrospective analysis was performed of the initial, relapse, and regular follow-up evaluations of patients with pelvic relapse following STLI alone from 1968 to the present for supradiaphragmatic Stage I-II Hodgkin's disease after pathologic staging (PS-laparotomy staging) and clinical staging (CS-no laparotomy staging). Following staging, which included bipedal lymphangiography, 482 patients (408 PS and 74 CS), were treated with STLI alone for supradiaphragmatic Stage I-II Hodgkin's disease. The actuarial freedom from relapse at 20 years was 75% in PS patients and 81% in CS patients. The actuarial pelvic failure at 20 years was 7% for PS patients and 3% for CS patients. Of the 29 patients with pelvic relapse, 97% (28 of 29) occurred within 5 years of treatment, including 1 patient who progressed during initial treatment. Pelvic relapse was most commonly initially identified by abnormalities involving patient symptoms (62%), physical examination (55%), erythrocyte sedimentation rate (48%), and bipedal lymphangiogram and/or abdominal radiograph (38%). Relapse was limited to previously unirradiated sites in 17 patients (58%). In addition to pelvic lymph node disease, 3 patients (10%) had involvement of bone, and 4 patients (14%) had bone marrow involvement. Following relapse, all patients were treated with chemotherapy (MOP[P], MOP[P]/ABV[D], ABVD, or PAVe) and 19 of 29 patients received involved field consolidative irradiation. Twenty-one of 29 (72%) remained relapse free at the time of last follow-up evaluation, including 15 of 19 (79%) treated with combined therapy. Eight patients experienced a second relapse despite salvage therapy, and all eight expired with recurrent Hodgkin's disease. Two patients died of complications related to prior treatment. Therefore, the actuarial risk of death at 20 years associated with pelvic failure in the entire cohort of 482 patients was 2%. Pelvic relapse occurred in 7% of patients following STLI alone and was effectively diagnosed by regular follow-up, which included a combination of patient history, physical examination, and radiographic laboratory evaluation. Seventy-two percent of patients remained relapse free following salvage treatment, which included chemotherapy, resulting in an overall survival rate associated with pelvic control of 98%. This approach, therefore, spared the majority of patients the long-term risks associated with pelvic irradiation and/or chemotherapy, such as infertility, but maintained an excellent prognosis.


Social support, life stress, pain and emotional adjustment to advanced breast cancer.
The purpose of this study was to examine relationships between emotional adjustment to advanced breast cancer, pain, social support, and life stress. The cross-sectional sample was compromised of 102 women with metastatic and/or recurrent breast cancer who were recruited into a randomized psychosocial intervention study. All women completed baseline questionnaires assessing demographic and medical variables, social support, life stress, pain, and mood disturbance. Three types of social support were assessed: (1) number of persons in support system; (2) positive support; and (3) aversive support. On the Profile of Mood States (POMS) total score, we found significant interactions between life stress and social support; having more people in the patient's support system was associated with less mood disturbance, but only among patients who had undergone greater life stress. Also, aversive social contact was significantly related to total mood disturbance (POMS), and having more aversive social contact was particularly associated with total mood disturbance (POMS) among patients who had undergone greater life stress. Pain intensity was associated with greater total life stress, and was not significantly related to social support. These results are consistent with the 'buffering hypothesis' that social support may shield women with metastatic breast cancer from the effects of previous life stress on their emotional adjustment; however, aversive support may be an additional source of life stress associated with emotional distress. Also, pain is greater among women with greater life stress, regardless of social support.


Identification of signatures of selection for intramuscular fat and backfat thickness in two Duroc populations.
Intramuscular fat (IMF) content is an important trait affecting the quality of pork. Two Duroc populations, one under positive selection for IMF and the other selected for decreased backfat but under stabilizing selection for IMF, were used to identify signatures of selection associated with IMF using 60,000 single-nucleotide polymorphism data. The effects of selection were analyzed between 2 lines or groups representing selected and control animals within each population using a discriminant analysis of principal components and Wright's fixation index (FST). Moreover, extended haplotype homozygosity-based approaches were used to examine the changes in haplotype frequency due to recent selection. Each statistical method identified 10–20 selection signatures. A few haplotype-based signatures of selection agreed with results from a genome-wide association study (GWAS), while FST measures showed a better agreement with GWAS results. Agreement of marker-trait associations and signatures of selection was limited, and further examination will be necessary to understand the effect of selection on IMF and why some regions identified by GWAS did not appear to respond to the selection practiced. The genes in 21 consensus selection signatures were examined. Several genes with an effect on overall fatness were identified, but further research is needed to assess whether or not some of them could have a specific effect on IMF.


Conference Report: Eighth Annual AACR International Conference on Frontiers in Cancer Prevention Research.
The Eighth Annual Frontiers in Cancer Prevention Research meeting was held in Houston, Texas, in November 2009. This report highlights significant presentations that advance the fields of chemoprevention, clinical trial recruitment and retention, cancer screening including optical imaging, energy balance, and nutritional epidemiology, and health communications and decision making. In findings from the randomized Reduction by Dutasteride of Prostate Cancer Events trial, dutasteride reduced the risk of biopsy-detectable prostate cancer in high-risk men by 23% compared with placebo. Important clues about the dosing and window of susceptibility for supplementation with choline, vitamin D, and folate were revealed from epigenetic research that has implications for future nutritional epidemiology research. Noninvasive optical imaging techniques using endoscopic ultrasound and autofluorescence for the early detection of cancers in the lung, pancreas, and oral cavity are being studied. The report also addresses the challenges of promoting cancer prevention. Understanding how individuals process risk information and make sustained behavior changes and the effect of socioeconomic status on health disparities were identified as critical areas of research. This multidisciplinary research meeting of basic, clinical, and behavioral scientists and epidemiologists continues to play a major role in identifying the research priority areas of cancer prevention, elucidating new mechanisms of carcinogenesis for targeted chemoprevention therapies and delivering a comprehensive strategy for engaging individuals in the unifying goal to reduce cancer incidence.


A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity.
Human in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology.


Interaction between the RGS domain of RGS4 with G protein alpha subunits mediates the voltage-dependent relaxation of the G protein-gated potassium channel.
1. In native cardiac myocytes, there is a time dependence to the G protein-gated inwardly rectifying K(+) (K(G)) channel current during voltage steps that accelerates as the concentration of acetylcholine is increased. This phenomenon has been called 'relaxation' and is not reproduced in the reconstituted Kir3.1/Kir3.4 channel in Xenopus oocytes. We have shown that RGS4, a regulator of G protein signalling, restores relaxation to the reconstituted Kir3.1/Kir3.4 channel. In this study, we examined the mechanism of this phenomenon by expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and mutants of RGS4 in Xenopus oocytes. 2. RGS4 restored relaxation to K(G) channels activated by the pertussis toxin (PTX)-sensitive G protein-coupled m(2)-muscarinic receptor but not to those activated by the G(s) protein-coupled beta(2)-adrenergic receptor. 3. RGS4 induced relaxation not only in heteromeric K(G) channels composed of Kir3.1 and Kir3.4 but also in homomeric assemblies of either an active mutant of Kir3.1 (Kir3.1/F137S) or an isoform of Kir3.2 (Kir3.2d). 4. Truncation mutants of RGS4 showed that the RGS domain itself was essential to reproduce the effect of wild-type RGS4 on the K(G) channel. 5. The mutation of residues in the RGS domain which interact with the alpha subunit of the G protein (G(alpha)) impaired the effect of RGS4. 6. This study therefore shows that interaction between the RGS domain and PTX-sensitive G(alpha) subunits mediates the effect of RGS4 on the agonist concentration-dependent relaxation of K(G) channels.


[Reflections on smoking intervention opportunities in Latin America].
The tobacco epidemic is a huge public health problem affecting all Latin American countries. These countries have the characteristic of a wide base of the pyramidal population structure, with many young people who have been natural targets for the tobacco industry. Therefore in this region there is an urgent need to establish prevention and treatment strategies capable to reduce incidence and prevalence of tobacco smoking in the population and as a consequence modify the health effects of tobacco consumption. Treating nicotine addiction requires intervention strategies really effective to reduce the number of current smokers as well as giving them tools to avoid relapse and maintain abstinence. Currently poor and vulnerable groups, which represent 44% of the total population in Latin America, are the most susceptible to suffer the health consequences of smoking since they show the highest prevalence rates and have little or no access to health services. The Framework Convention on Tobacco Control (FCTC) and MPOWER, both strategies proposed by the World Health Organization against tobacco, define clear goals to achieve their purposes, nevertheless they are limited by the economic and human resources assigned by each country. The scientific community involved in the fight against tobacco must think about new ways to place this issue in the political agenda so there will be more opportunities to increase the resources and therefore to reduce tobacco consumption in all the groups of the population. In the fight against tobacco, this is the right time to consider primarily issues related to equity and social justice.


Differential decay of intact and defective proviral DNA in HIV-1-infected individuals on suppressive antiretroviral therapy.
BACKGROUNDThe relative stabilities of the intact and defective HIV genomes over time during effective antiretroviral therapy (ART) have not been fully characterized.METHODSWe used the intact proviral DNA assay (IPDA) to estimate the rate of change of intact and defective proviruses in HIV-infected adults on ART. We used linear spline models with a knot at seven years and a random intercept and slope up to the knot. We estimated the influence of covariates on rates of change.RESULTSWe studied 81 individuals for a median of 7.3 (IQR 5.9-9.6) years. Intact genomes declined more rapidly from initial suppression through seven years (15.7% per year decline; 95% CI -22.8%, -8.0%) and more slowly after seven years (3.6% per year; 95% CI -8.1%, +1.1%). The estimated half-life of the reservoir was 4.0 years (95% CI 2.7-8.3) until year seven and 18.7 years (95% CI 8.2-infinite) thereafter. There was substantial variability between individuals in the rate of decline until year seven. Intact provirus declined more rapidly than defective provirus (P < 0.001) and showed a faster decline in individuals with higher CD4+ T cell nadirs.CONCLUSIONThe biology of the replication-competent (intact) reservoir differs from that of the replication-incompetent (non-intact) pool of proviruses. The IPDA will likely be informative when investigating the impact of interventions targeting the reservoir.FUNDINGDelaney AIDS Research Enterprise, UCSF/Gladstone Institute of Virology & Immunology CFAR, CFAR Network of Integrated Systems, amfAR Institute for HIV Cure Research, I4C and Beat-HIV Collaboratories, Howard Hughes Medical Institute, Gilead Sciences, Bill and Melinda Gates Foundation.


Longer term clinical and economic benefits of offering acupuncture care to patients with chronic low back pain.
To test whether patients with persistent non-specific low back pain, when offered access to traditional acupuncture care alongside conventional primary care, gained more long-term relief from pain than those offered conventional care only, for equal or less cost. Safety and acceptability of acupuncture care to patients, and the heterogeneity of outcomes were also tested. A pragmatic, two parallel group, randomised controlled trial. Patients in the experimental arm were offered the option of referral to the acupuncture service comprising six acupuncturists. The control group received usual care from their general practitioner (GP). Eligible patients were randomised in a ratio of 2:1 to the offer of acupuncture to allow between-acupuncturist effects to be tested. Three non-NHS acupuncture clinics, with referrals from 39 GPs working in 16 practices in York, UK. Patients aged 18-65 years with non-specific low back pain of 4-52 weeks' duration, assessed as suitable for primary care management by their general practitioner. The trial protocol allowed up to ten individualised acupuncture treatments per patient. The acupuncturist determined the content and the number of treatments according to patient need. The Short Form 36 (SF-36) Bodily Pain dimension (range 0-100 points), assessed at baseline, and 3, 12 and 24 months. The study was powered to detect a 10-point difference between groups at 12 months post-randomisation. Cost--utility analysis was conducted at 24 months using the EuroQoL 5 Dimensions (EQ-5D) and a preference-based single index measure derived from the SF-36 (SF-6D). Secondary outcomes included the McGill Present Pain Index (PPI), Oswestry Pain Disability Index (ODI), all other SF-36 dimensions, medication use, pain-free months in the past year, worry about back pain, satisfaction with care received, and safety and acceptability of acupuncture care. A total of 159 patients were in the 'acupuncture offer' arm and 80 in the 'usual care' arm. All 159 patients randomised to the offer of acupuncture care chose to receive acupuncture treatment, and received an average of eight acupuncture treatments within the trial. Analysis of covariance, adjusting for baseline score, found an intervention effect of 5.6 points on the SF-36 Pain dimension [95% confidence interval (CI) -1.3 to 12.5] in favour of the acupuncture group at 12 months, and 8 points (95% CI 0.7 to 15.3) at 24 months. No evidence of heterogeneity of effect was found for the different acupuncturists. Patients receiving acupuncture care did not report any serious or life-threatening events. No significant treatment effect was found for any of the SF-36 dimensions other than Pain, or for the PPI or the ODI. Patients receiving acupuncture care reported a significantly greater reduction in worry about their back pain at 12 and 24 months compared with the usual care group. At 24 months, the acupuncture care group was significantly more likely to report 12 months pain free and less likely to report the use of medication for pain relief. The acupuncture service was found to be cost-effective at 24 months; the estimated cost per quality-adjusted (QALY) was 4241 pounds sterling (95% CI 191 pounds sterling to 28,026 pounds sterling) using the SF-6D scoring algorithm based on responses to the SF-36, and 3598 pounds sterling (95% CI 189 pounds sterling to 22,035 pounds sterling) using the EQ-5D health status instrument. The NHS costs were greater in the acupuncture care group than in the usual care group. However, the additional resource use was less than the costs of the acupuncture treatment itself, suggesting that some usual care resource use was offset. Traditional acupuncture care delivered in a primary care setting was safe and acceptable to patients with non-specific low back pain. Acupuncture care and usual care were both associated with clinically significant improvement at 12- and 24-month follow-up. Acupuncture care was significantly more effective in reducing bodily pain than usual care at 24-month follow-up. No benefits relating to function or disability were identified. GP referral to a service providing traditional acupuncture care offers a cost-effective intervention for reducing low back pain over a 2-year period. Further research is needed to examine many aspects of this treatment including its impact compared with other possible short-term packages of care (such as massage, chiropractic or physiotherapy), various aspects of cost-effectiveness, value to patients and implementation protocols.


Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells.
In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.


Convergent evolution of alternative developmental trajectories associated with diapause in African and South American killifish.
Annual killifish adapted to life in seasonally ephemeral water-bodies exhibit desiccation resistant eggs that can undergo diapause, a period of developmental arrest, enabling them to traverse the otherwise inhospitable dry season. Environmental cues that potentially indicate the season can govern whether eggs enter a stage of diapause mid-way through development or skip this diapause and instead undergo direct development. We report, based on construction of a supermatrix phylogenetic tree of the order Cyprinodontiformes and a battery of comparative analyses, that the ability to produce diapause eggs evolved independently at least six times within African and South American killifish. We then show in species representative of these lineages that embryos entering diapause display significant reduction in development of the cranial region and circulatory system relative to direct-developing embryos. This divergence along alternative developmental pathways begins mid-way through development, well before diapause is entered, during a period of purported maximum developmental constraint (the phylotypic period). Finally, we show that entering diapause is accompanied by a dramatic reduction in metabolic rate and concomitant increase in long-term embryo survival. Morphological divergence during the phylotypic period thus allows embryos undergoing diapause to conserve energy by shunting resources away from energetically costly organs thereby increasing survival chances in an environment that necessitates remaining dormant, buried in the soil and surrounded by an eggshell for much of the year. Our results indicate that adaptation to seasonal aquatic environments in annual killifish imposes strong selection during the embryo stage leading to marked diversification during this otherwise conserved period of vertebrate development.


Dosing Recommendations of Aripiprazole Depot with Strong Cytochrome P450 3A4 Inhibitors: A Relapse Risk.
A 50-year-old male patient with comorbid human immunodeficiency virus developed a relapse of bipolar disorder after a switch from oral aripiprazole 10 mg/day to intramuscular aripiprazole depot 200 mg every 28 days plus oral aripiprazole 5 mg/day. The patient was concomitantly taking lopinavir, saquinavir, ritonavir, silybum marianum extract, and omeprazole. Only 1 week after the switch, the patient developed mood swings, irritability, depressive mood, and lack of drive. The oral aripiprazole was increased again to stabilize the patient. The measured trough drug concentration of aripiprazole was low and may be correlated to the relapse. When oral aripiprazole was increased back to 10 mg again, the depressive symptoms subsided. The dose of the next depot injection was increased to 300 mg and that of oral aripiprazole decreased back to 5 mg/day. Because trough drug concentrations were still low after 28 days, the depot dose was increased to 400 mg every 28 days, which is double that recommended in the prescriber's information. Two months after the initial switch from oral to intramuscular aripiprazole, the patient's mood stabilized on aripiprazole depot 400 mg every 28 days. More clinical data, especially regarding the pharmacokinetic drug interactions of aripiprazole depot are needed to improve dosing recommendations, and prevent relapses or adverse drug events. Genetic polymorphisms may play an important role in the clinical relevance of drug interactions concerning aripiprazole depot.


Modeling thrombosis in silico: Frontiers, challenges, unresolved problems and milestones.
Hemostasis is a complex physiological mechanism that functions to maintain vascular integrity under any conditions. Its primary components are blood platelets and a coagulation network that interact to form the hemostatic plug, a combination of cell aggregate and gelatinous fibrin clot that stops bleeding upon vascular injury. Disorders of hemostasis result in bleeding or thrombosis, and are the major immediate cause of mortality and morbidity in the world. Regulation of hemostasis and thrombosis is immensely complex, as it depends on blood cell adhesion and mechanics, hydrodynamics and mass transport of various species, huge signal transduction networks in platelets, as well as spatiotemporal regulation of the blood coagulation network. Mathematical and computational modeling has been increasingly used to gain insight into this complexity over the last 30 years, but the limitations of the existing models remain profound. Here we review state-of-the-art-methods for computational modeling of thrombosis with the specific focus on the analysis of unresolved challenges. They include: a) fundamental issues related to physics of platelet aggregates and fibrin gels; b) computational challenges and limitations for solution of the models that combine cell adhesion, hydrodynamics and chemistry; c) biological mysteries and unknown parameters of processes; d) biophysical complexities of the spatiotemporal networks' regulation. Both relatively classical approaches and innovative computational techniques for their solution are considered; the subjects discussed with relation to thrombosis modeling include coarse-graining, continuum versus particle-based modeling, multiscale models, hybrid models, parameter estimation and others. Fundamental understanding gained from theoretical models are highlighted and a description of future prospects in the field and the nearest possible aims are given.


First Report of Orange Rust Caused by Puccinia kuehnii in Sugarcane in Louisiana.
In June 2012, lesions typical of rust were observed on sugarcane cultivar Ho 05-961 (a complex hybrid of Saccharum L. spp.) on a farm near Schriever, Louisiana. Incidence and severity of disease symptoms were low. Two types of pustules were observed on leaves of the infected plants. One pustule type was reddish-brown in color turning brown with age, characteristic of brown rust which has been observed in Louisiana since 1979 (2). The other pustule type was orange and did not turn brown with age. Urediniospore samples from the two pustule types were collected. The morphology of the urediniospores from the reddish-brown pustules was consistent with that described for Puccinia melanocephala Syd. & Syd., the fungus that causes brown rust of sugarcane, while the morphology of the urediniospores from the orange pustules was consistent with those described for P. kuehnii E.J. Butler, the causal organism of orange rust of sugarcane (3). Telia and teliospores were not observed. The identity of the two species of Puccinia causing the brown and orange rust lesions was verified using the species-specific quantitative PCR assays (1). Two DNA samples extracted from the pustules identified as P. kuehnii were independently subjected to PCR amplification using primers Pk1F and Pk1R (1) to yield a product from the rDNA that was then bidirectionally sequenced using the same primers. The resulting 480-nt sequences were identical to each other, and a BLAST search of GenBank revealed 100% identity to 19 previously reported isolates of P. kuehnii but not more than 89% similarity to any isolate of P. melanocephala (4). To our knowledge, this is the first report of orange rust in Louisiana. In the 4 months following the detection of orange rust, observations of the disease have been limited to Ho 05-961. Seed cane increase plots of this newly released cultivar were surveyed, and orange rust symptoms and urediniospores were detected in 17 of 38 (45%) fields. The incidence and severity of the disease remained low, and the distribution appeared to be limited to the southern portion of the Louisiana sugarcane production area. References: (1) N. C. Glynn et al. Plant Pathol. 59:703, 2010. (2) H. Koike. Plant Dis. 64:226, 1980. (3) C. C. Ryan et al. Page 189 in: Diseases of Sugarcane: Major Diseases. C. Ricaud et al., eds. Elsevier, Amsterdam, 1989. (4) E. V. Virtudazo et al. Mycoscience 42:447, 2001.


Inverting chaos: Extracting system parameters from experimental data.
Given a set of experimental or numerical chaotic data and a set of model differential equations with several parameters, is it possible to determine the numerical values for these parameters using a least-squares approach, and thereby to test the model against the data? We explore this question (a) with simulated data from model equations for the Rossler, Lorenz, and pendulum attractors, and (b) with experimental data produced by a physical chaotic pendulum. For the systems considered in this paper, the least-squares approach provides values of model parameters that agree well with values obtained in other ways, even in the presence of modest amounts of added noise. For experimental data, the "fitted" and experimental attractors are found to have the same correlation dimension and the same positive Lyapunov exponent. (c) 1996 American Institute of Physics.


Syphilitic uveitis and optic neuritis in Sydney, Australia.
To describe ocular syphilis presentations to a tertiary referral eye hospital over a 5-year period and to document HIV coinfection frequency. A retrospective chart review was conducted of consecutive ocular syphilis presentations to Sydney Eye Hospital from 2007 to 2012. Inclusion criteria were positive syphilis serology, ocular inflammation on clinical examination and appropriate syphilis treatment. Outcome measures were clinical features at presentation and best-corrected visual acuity (BCVA) at interval follow-up. Thirty-seven eyes of 25 patients were included in the series. Patients were predominantly male (92.0%, p<0.05) with mean age 43.7±14.0 years. Eight (32.0%) patients had confirmed HIV coinfection, three newly diagnosed with HIV. Twelve (32.4%) eyes demonstrated anterior segment involvement with anterior uveitis. Twenty-five (67.6%) eyes demonstrated posterior segment involvement, including panuveitis, acute syphilitic posterior placoid chorioretinitis, retinitis, necrotising retinitis, punctate retinitis and optic neuritis. There was a significant improvement in BCVA for involved eyes (p<0.05) at 1 month and 2-3 months follow-up. The clinical findings of 37 eyes with ocular syphilis demonstrated a broad spectrum of clinical manifestations. Rates of HIV coinfection were high, with patients exhibiting both anterior and posterior segment inflammation. Visual outcome improved following syphilis treatment.


Bilateral thoracoscopic sympathectomy for primary hyperhydrosis: a review of 335 cases.
The goal of this retrospective study was to evaluate the outcomes of bilateral video-assisted thoracoscopic sympathectomy for primary hyperhydrosis. Between January 2007 and December 2011, a total of 335 patients (192 male, 143 female, mean age 28.3 years) who underwent bilateral thoracoscopic sympathectomy for primary hyperhydrosis were reviewed retrospectively. Hyperhydrosis occurred in the palmar and axillary region in 175 (52.23%) patients, in only the palmar region in 52 (15.52%), in the craniofacial region in 44 (13.13%), in only the axillary region in 42 (12.53%), and in the palmar and pedal regions in 22 (6.56%) patients. Bilateral thoracoscopic sympathectomy was performed in all patients. The mean follow-up period was 24 (6-48) months. The initial cure rate was 95% and the initial satisfaction rate was 93%. There was no mortality in this study. The complication rate was 15.82% in 53 patients. Video-assisted thoracoscopic sympathectomy for the treatment of primary hyperhydrosis was effective, with low rates of morbidity and mortality. Despite the appearance of postoperative complications, such as compensatory sweating, patient satisfaction with the procedure was high and their quality of life improved.


Evaluating the impact of the weather conditions on the influenza propagation.
Predicting the details of how an epidemic evolves is highly valuable as health institutions need to better plan towards limiting the infection propagation effects and optimizing their prediction and response capabilities. Simulation is a cost- and time-effective way of predicting the evolution of the infection as the joint influence of many different factors: interaction patterns, personal characteristics, travel patterns, meteorological conditions, previous vaccination, etc. The work presented in this paper extends EpiGraph, our influenza epidemic simulator, by introducing a meteorological model as a modular component that interacts with the rest of EpiGraph's modules to refine our previous simulation results. Our goal is to estimate the effects of changes in temperature and relative humidity on the patterns of epidemic influenza based on data provided by the Spanish Influenza Sentinel Surveillance System (SISSS) and the Spanish Meteorological Agency (AEMET). Our meteorological model is based on the regression model developed by AB and JS, and it is tuned with influenza surveillance data obtained from SISSS. After pre-processing this data to clean it and reconstruct missing samples, we obtain new values for the reproduction number of each urban region in Spain, every 10 minutes during 2011. We simulate the propagation of the influenza by setting the date of the epidemic onset and the initial influenza-illness rates for each urban region. We show that the simulation results have the same propagation shape as the weekly influenza rates as recorded by SISSS. We perform experiments for a realistic scenario based on actual meteorological data from 2010-2011, and for synthetic values assumed under simplified predicted climate change conditions. Results show that a diminishing relative humidity of 10% produces an increment of about 1.6% in the final infection rate. The effect of temperature changes on the infection spread is also noticeable, with a decrease of 1.1% per extra degree. Using a tool like ours could help predict the shape of developing epidemics and its peaks, and would permit to quickly run scenarios to determine the evolution of the epidemic under different conditions. We make EpiGraph source code and epidemic data publicly available.


Epigenetic activation of the Foxa2 gene is required for maintaining the potential of neural precursor cells to differentiate into dopaminergic neurons after expansion.
Dysregulation of forkhead box protein A2 (Foxa2) expression in fetal ventral mesencephalon (VM)-derived neural precursor cells (NPCs) appears to be associated with the loss of their potential to differentiate into dopaminergic (DA) neurons after mitogenic expansion in vitro, hindering their efficient use as a transplantable cell source. Here, we report that epigenetic activation of Foxa2 in VM-derived NPCs by inducing histone hyperacetylation rescues the mitogenic-expansion-dependent decrease of differentiation potential to DA neurons. The silencing of Foxa2 gene expression after expansion is accompanied by repressive histone modifications, including hypoacetylation of histone H3 and H4 and trimethylation of H3K27 on the Foxa2 promoter, as well as on the global level. In addition, histone deacetylase 7 (HDAC7) is highly expressed during differentiation and recruited to the Foxa2 promoter. Induction of histone acetylation in VM-derived NPCs by either knockdown of HDAC7 or treatment with the HDAC inhibitor apicidin upregulates Foxa2 expression via hyperacetylation of H3 and a decrease in H3K27 trimethylation on the promoter regions, leading to the expression of DA neuron developmental genes and enhanced differentiation of DA neurons. These effects are antagonized by the expression of shRNAs specific for Foxa2 but enhanced by shRNA for HDAC7. Collectively, these findings indicate that loss of differentiation potential of expanded VM-derived NPCs is attributed to a decrease in Foxa2 expression and suggest that activation of the endogenous Foxa2 gene by epigenetic regulation might be an approach to enhance the generation of DA neurons.


Highly syntenic and yet divergent: a tale of two Theilerias.
The published genomic sequences of the two major host-transforming Theileria species of cattle represent a rich resource of information that has allowed novel bioinformatic and experimental studies into these important apicomplexan parasites. Since their publication in 2005, the genomes of T. annulata and T. parva have been utilised for a diverse range of applications, ranging from candidate antigen discovery to the identification of genetic markers for population analysis. This has led to advancements in the quest for a sub-unit vaccine, while providing a greater understanding of variation among parasite populations in the field. The unique ability of these Theileria species to induce host cell transformation is the subject of considerable scientific interest and the availability of full genomic sequences has provided new insights into this area of research. This article reviews the data underlying published comparative analyses, focussing on the general features of gene expression, the major Tpr/Tar multi-copy gene family and a re-examination of the predicted macroschizont secretome. Codon usage between the Theileria species is reviewed in detail, as this underpins ongoing comparative studies investigating selection at the intra- and inter-species level. The TashAT/TpshAT family of genes, conserved between T. annulata and T. parva, encodes products targeted to the host nucleus and has been implicated in contributing to the transformed bovine phenotype. Species-specific expansion and diversification at this critical locus is discussed with reference to the availability, in the near future, of genomic datasets which are based on non-transforming Theileria species.


Cardiac output during exercise in patients with COPD.
Cardiac output response was examined to determine its relationship to exercise and arterial hypoxia, pulmonary hemodynamics, and the clinical/lung mechanics profile of patients with chronic obstructive pulmonary disease (COPD). Twenty patients with COPD were examined on the treadmill at rest and during steady state exercise at the highest workload comfortably tolerable to the subjects (60-70% VO2 max). In eight patients, the exercise cardiac output was less than 80% of predicted, while in the remainder (n = 12), the cardiac output response was normal (greater than 80% of predicted). It was concluded that hyperinflation and severe expiratory airflow limitation may be important determinants of the low exercise cardiac output response in some patients with COPD.


Clinical, physiological, and histological features in a kindred with the T3271C melas mutation.
The majority of patients with MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) have an A-->G mutation at nucleotide 3243 in mitochondrial transfer (t)RNA. To date there have only been 10 reported cases of MELAS syndrome in patients with a T-->C mutation at position 3271 of mitochondrial tRNA. Although many of the clinical features are similar between patients with these different mutations, it appears that the age at onset is later for the 3271 mutation. This report provides information from a North American kindred with the 3271 mutation (n = 6 proven; n = 2 probable; n = 3 possible) that adds clinical, physiological, histological, and molecular information to the pool of information on this rare disorder. Many of these features were similar to previous reports of both 3243 and 3271 patients. We conclude that the phenotypic expression of these different mutations are similar, but the age of onset for 3271 patients is later than for 3243 patients.


Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK.
The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk(-/-) HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk(-/-) HSC expansion is dependent on THPO, and 12-wk-old Lnk(-/-)Thpo(-/-) mice have 65-fold fewer LT-HSCs than Lnk(-/-) mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk(-/-) mice also proved THPO-dependent.


The mechanism of carcinogenesis by the neutral fraction of cigarette smoke condensate.
Sixteen groups, each of 50 Swiss female SPF mice, were treated thrice weekly with various combinations of 3,4-benzopyrene (BP) and/or the neutral fraction of cigarette smoke (NF) in acetone applied to the skin. Some groups received one carcinogen, some the other and some a mixture of the two. Skin tumour incidence rates were found to increase both with the dose of NF and with the dose of BP. With BP alone a threshold dose was found beyond which a very heavy incidence rate of malignant skin tumours occurred. After correction of the results for intercurrent deaths it was found that when NF and BP are applied together as a mixture they do not act independently in the production of malignant skin tumours but interact positively. This suggests that some of the components of NF act as cocarcinogens rather than as complete carcinogens. Treatment with NF appeared to increase the incidence of malignant lymphomas. The data were not suitable for deciding whether the various treatments influenced the rates of incidence of internal tumours of other types, for example, lung tumours.


Resilience in Non-Offending Mothers of Children Who Have Reported Experiencing Sexual Abuse.
Growing evidence shows that non-offending mothers experience significant loss and trauma following their child reporting experiencing sexual abuse. Maternal support offered to sexually abused children following disclosure can be a crucial factor in children's recovery. Although mothers are often seen as playing a central role in facilitating the recovery of their sexually abused child, there has been little focus on their own needs and profiles. This present study aimed to increase our understanding of the diversity of profiles of non-offending mothers of sexually abused children by exploring the differences in psychosocial traits of non-offending mothers (N = 68; age range 28-67 years) reporting higher and lower resilience. The mediating role of these psychosocial factors on the relationship between resilience and psychological distress will also be explored. Results found that non-offending mothers in the higher resilience group reported higher levels of positive reappraisal, self-compassion, social support, and significantly lower levels of psychological distress compared to non-offending mothers in the lower resilience group. Additionally, multiple mediation analysis indicated positive reappraisal, self-compassion, and social support to be significant mediators of the relationship between resilience and psychological distress. Increased levels of self-compassion and social support were found to be predictive of lowered psychological distress, while increased positive reappraisal predicted increased psychological distress. The findings of the present study provide support for the targeting of the psychosocial factors such as self-compassion, social support, and positive reappraisal in interventions for non-offending mothers in an effort to promote resilience.


GFRA1: A Novel Molecular Target for the Prevention of Osteosarcoma Chemoresistance.
The glycosylphosphatidylinositol-linked GDNF (glial cell derived neurotrophic factor) receptor alpha (GFRA), a coreceptor that recognizes the GDNF family of ligands, has a crucial role in the development and maintenance of the nervous system. Of the four identified GFRA isoforms, GFRA1 specifically recognizes GDNF and is involved in the regulation of proliferation, differentiation, and migration of neuronal cells. GFRA1 has also been implicated in cancer cell progression and metastasis. Recent findings show that GFRA1 can contribute to the development of chemoresistance in osteosarcoma. GFRA1 expression was induced following treatment of osteosarcoma cells with the popular anticancer drug, cisplatin and induction of GFRA1 expression significantly suppressed apoptosis mediated by cisplatin in osteosarcoma cells. GFRA1 expression promotes autophagy by activating the SRC-AMPK signaling axis following cisplatin treatment, resulting in enhanced osteosarcoma cell survival. GFRA1-induced autophagy promoted tumor growth in mouse xenograft models, suggesting a novel function of GFRA1 in osteosarcoma chemoresistance.


The PDZ domain of the SpoIVB serine peptidase facilitates multiple functions.
During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the sigma(K) regulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor sigma(K) in the outer mother cell chamber of the sporulating cell. This forms the basis of the sigma(K) checkpoint and ensures accurate sigma(K)-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for trans proteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-sigma(K) processing, and (iv) signaling of the second function of SpoIVB.


Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.
Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration.


Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules.
Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.


From here to infinity: sparse finite versus Dirichlet process mixtures in model-based clustering.
In model-based clustering mixture models are used to group data points into clusters. A useful concept introduced for Gaussian mixtures by Malsiner Walli et al. (Stat Comput 26:303-324, 2016) are sparse finite mixtures, where the prior distribution on the weight distribution of a mixture with K components is chosen in such a way that a priori the number of clusters in the data is random and is allowed to be smaller than K with high probability. The number of clusters is then inferred a posteriori from the data. The present paper makes the following contributions in the context of sparse finite mixture modelling. First, it is illustrated that the concept of sparse finite mixture is very generic and easily extended to cluster various types of non-Gaussian data, in particular discrete data and continuous multivariate data arising from non-Gaussian clusters. Second, sparse finite mixtures are compared to Dirichlet process mixtures with respect to their ability to identify the number of clusters. For both model classes, a random hyper prior is considered for the parameters determining the weight distribution. By suitable matching of these priors, it is shown that the choice of this hyper prior is far more influential on the cluster solution than whether a sparse finite mixture or a Dirichlet process mixture is taken into consideration.


Thermodynamic versus conformational metastability in fibril-forming lysozyme solutions.
The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation is a crucial aspect for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions of lysozyme at acidic pH and low ionic strength. The amyloid formation occurs after a long lag time and is preceded by the formation of oligomers, which seems to be off-pathway with respect to fibrillation. By measuring the osmotic isothermal compressibility and the collective diffusion coefficient of lysozyme in solution, we observe that the monomeric solution is kept in a thermodynamically metastable state by strong electrostatic repulsion, even in denaturing conditions. The measured repulsive interaction between monomers is satisfactorily accounted for by classical polyelectrolyte theory. Further, we observe a slow conformational change involving both secondary and tertiary structure, which drives the proteins toward a more hydrophobic conformation. Denatured proteins are driven out of metastability through conformational substates, which are kinetically populated and experience a lower activation energy for fibril formation. Thus, our results highlight the role of electrostatic repulsion, which hinders the aggregation of partially denatured proteins and operates as a gatekeeper favoring the association of those monomers whose conformation is capable of forming amyloid structure.